DESCRIPTION: (provided by the applicant): Tick-borne diseases are increasingly diagnosed in humans and animals. Some are due to the resurgence of previously known illnesses, like Rocky Mountain spotted fever (Walker 1995). but others are due to new, emerging pathogens. Among the spotted fever group (SFG) alone, 8 new human pathogens have been described in the last 15 years (Stenos et al. 1997; Nilsson et al. 1999; Fournier et al. 2000), but they also include viruses, ehilichias and Babesia (Dawson et al. 1991; Thomford et al. 1994; Bakken et al. 1994; Telford et al 1991). Novel, efficient, specific and environmentally acceptable methods that interfere with disease transmission by ticks are urgently needed. Using paratransgenic ticks that carry symbiotic prokaryotes expressing an antimicrobial substance, as has been achieved with the symbiote of the Chagas disease vector, Rhodnius prolixus (Durvasula et aL 1997), could offer a safe and effective way to reduce disease transmission by ticks. A major obstacle to accomplishing this goal has been the lack of culture systems for tick symbiotes. Our laboratory has the largest collection of tick cell lines. We have successfully used these to isolate tick-associated rickettsiae (Munderloh et al. 1998; Weller et al. 1998; Palmer et al. 1999; Simser et al. 2001a,b) from the Lone Star tick (MOAa), the Rocky Mountain wood tick (R. peacockli DAE100R), and the Castor Bean tick (Rmoreli T2). We have characterized these microbes by light and electron microscopy, by using specific antibodies, as well as by PCR and nucleotide sequence analysis of 165 rDNA and other key genes. We are now in the process of defining the cultures to facilitate genetic manipulation of the symbiotes. Ourlong-termaim is the stabletransformation of Rickettsiapeacockii with cecropinA. an insect ponn gene (Hultmark et al. 1983). Infection of ticks with the transformed rickettsia, and interference with pathogen transmission. We plan to target the non-functional rompA gene of R. peacockli as a site for homologous transformation, avoiding deleterious effects associated with disruption of a vital gene, e.g. the rpoB gene (troyer et al. 1999). We will take the recent advances in successful transformation of insect-borne rickettsiae as a guide (Rachek et al. 1998; Troyer et al. 1999). and also apply transposome technology (Epicentre). Specifically, we will 1. optimize culture conditions for production of R. peacockii in tick cell culture, exanine its behavior in tick and mammalian cell culture by light and electron microscopy. 2. We will analyze cultured R. peacockii in ticks in terms of tissue tropism and transstadial/transovanal passage, and sensitivity to Cecropin A. Finally, we will work towards 3. stable transformation of R. peacockii with cecropinA. We will then test the transfonnants for antimicrobial activity in vitro and in ticks, and characterize them by sequence analysis.